Concentration determination in a diffusion barrier layer

ABSTRACT

The present invention relates to improved electrochemical biosensor strips and methods for determining the concentration of an analyte in a sample. By selectively measuring a measurable species residing in a diffusion barrier layer, to the substantial exclusion of the measurable species residing exterior to the diffusion barrier layer, measurement errors introduced by sample constituents, such as red blood cells, and manufacturing variances may be reduced.

REFERENCE TO RELATED APPLICATIONS

This application is a continuation of divisional U.S. application Ser.No. 13/669,674 entitled “Concentration Determination in A DiffusionBarrier Layer” filed Nov. 6, 2012, which is a divisional of U.S.application Ser. No. 11/734,251 entitled “Concentration Determination inA Diffusion Barrier Layer” filed Apr. 11, 2007, now U.S. Pat. No.8,317,988 issued on Nov. 27, 2012, which was a continuation ofPCT/US2005/036806 entitled “Concentration Determination in A DiffusionBarrier Layer” filed Oct. 12, 2005, which was published in English andclaimed the benefit of U.S. Provisional Application No. 60/617,889entitled “Concentration Determination in A Diffusion Barrier Layer”filed Oct. 12, 2004 and of U.S. Provisional Application No. 60/655,180entitled “Concentration Determination in A Diffusion Barrier Layer”filed Feb. 22, 2005. Each of these applications is incorporated byreference in its entirety.

BACKGROUND

In monitoring medical conditions and the response of patients totreatment efforts, it is desirable to use analytical methods that arefast, accurate, and convenient for the patient. Electrochemical methodshave been useful for quantifying certain analytes in body fluids,particularly in blood samples. Typically, these biological analytes,such as glucose, undergo redox reactions when in contact with specificenzymes. The electric current generated by such redox reactions may becorrelated with the concentration of the biological analyte in thesample.

Tiny electrochemical cells have been developed that provide patients thefreedom to monitor blood analyte concentrations without the need of ahealthcare provider or clinical technician. Typical patient-operatedelectrochemical systems utilize a disposable sensor strip with adedicated measurement device containing the necessary circuitry andoutput systems. For analysis, the measurement device is connected to thedisposable electrochemical sensor strip containing the electrodes andreagents to measure the analyte concentration in a sample that isapplied to the strip.

The most common of these miniature electrochemical systems are glucosesensors that provide measurements of blood glucose levels. Ideally, aminiature sensor for glucose should provide accurate readings of bloodglucose levels by analyzing a single drop of whole blood, typically from1-15 microliters (μL).

In a typical analytical electrochemical cell, the oxidation or reductionhalf-cell reaction involving the analyte produces or consumes electrons,respectively. This electron flow can be measured, provided the electronscan interact with a working electrode that is in contact with the sampleto be analyzed. The electrical circuit is completed through a counterelectrode that is also in contact with the sample. A chemical reactionalso occurs at the counter electrode, and this reaction is of theopposite type (oxidation or reduction) relative to the type of reactionat the working electrode. Thus, if oxidation occurs at the workingelectrode, reduction occurs at the counter electrode. See, for example,Fundamentals Of Analytical Chemistry, 4^(th) Edition, D. A. Skoog and D.M. West; Philadelphia: Saunders College Publishing (1982), pp 304-341.

Some conventional miniaturized electrochemical systems include a truereference electrode. In these systems, the true reference electrode maybe a third electrode that provides a non-variant reference potential tothe system, in addition to the working and counter electrodes. Whilemultiple reference electrode materials are known, a mixture of silver(Ag) and silver chloride (AgCl) is typical. The materials that providethe non-variant reference potential, such as a mixture of silver andsilver chloride, are separated, by their insolubility or other means,from the reaction components of the analysis solution.

In other miniature electrochemical systems, a combinationcounter/reference electrode is employed. These electrochemical sensorstrips are typically two electrode systems having a working electrodeand a counter/reference electrode. The combined counter/referenceelectrode is possible when a true reference electrode also is used asthe counter electrode.

Because they are true reference electrodes, counter/reference electrodesare typically mixtures of silver (Ag) and silver chloride (AgCl), whichexhibit stable electrochemical properties due to the insolubility of themixture in the aqueous environment of the analysis solution. Since theratio of Ag to AgCl does not significantly change during transient use,the potential of the electrode is not significantly changed.

An electrochemical sensor strip is typically made by coating a reagentlayer onto the conductor surface of an analysis strip. To facilitatemanufacturing, the reagent layer may be coated as a single layer ontoall of the electrodes.

The reagent layer may include an enzyme for facilitating the oxidationor reduction reaction of the analyte, as well as any mediators or othersubstances that assist in the transfer of electrons between the analytereaction and the conductor surface. The reagent layer also may include abinder that holds the enzyme and mediator together, thus allowing themto be coated onto the electrodes.

Whole blood (WB) samples contain red blood cells (RBC) and plasma. Theplasma is mostly water, but contains some proteins and glucose.Hematocrit is the volume of the RBC constituent in relation to the totalvolume of the WB sample and is often expressed as a percentage. Wholeblood samples generally have hematocrit percentages ranging from 20 to60%, with ˜40% being the average.

One of the drawbacks of conventional electrochemical sensor stripsutilized to measure the glucose concentration in WB is referred to asthe “hematocrit effect.” The hematocrit effect is caused by RBC blockingthe diffusion of the mediator or other measurable species to theconductor surface for measurement. Because the measurement is taken forthe same time period each time a sample is tested, blood samples havingvarying concentrations of RBC can cause inaccuracies in the measurement.This is true because the sensor cannot distinguish between a lowermeasurable species concentration and a higher measurable speciesconcentration where the RBC interfere with diffusion of the measurablespecies to the conductor surface. Thus, variances in the concentrationof RBC in the WB sample result in inaccuracies (the hematocrit effect)in the glucose reading.

If WB samples containing identical glucose levels, but havinghematocrits of 20, 40, and 60%, are tested, three different glucosereadings will be reported by a conventional system that is based on oneset of calibration constants (slope and intercept, for instance). Eventhough the glucose concentrations are the same, the system will reportthat the 20% hematocrit sample contains more glucose than the 60%hematocrit sample due to the RBC interfering with diffusion of themeasurable species to the conductor surface.

Conventional systems are generally configured to report glucoseconcentrations assuming a 40% hematocrit content for the WB sample,regardless of the actual hematocrit content in the blood sample. Forthese systems, any glucose measurement performed on a blood samplecontaining less or more than 40% hematocrit will include some inaccuracyattributable to the hematocrit effect.

Conventional methods of reducing the hematocrit effect for amperometricsensors include the use of filters, as disclosed in U.S. Pat. Nos.5,708,247 and 5,951,836; reversing the potential of the read pulse, asdisclosed in WO 01/57510; and by methods that maximize the inherentresistance of the sample, as disclosed in U.S. Pat. No. 5,628,890. Whileeach of these methods balance various advantages and disadvantages, noneare ideal.

As can be seen from the above description, there is an ongoing need forimproved devices and methods for determining the concentration ofbiological analytes, including glucose. The devices and methods of thepresent invention may decrease the error introduced by the hematocritand other effects in WB samples.

SUMMARY

In one aspect, an electrochemical sensor strip is provided that includesa base and first and second electrodes on the base. The first electrodeincludes at least one first layer on a first conductor, where the firstlayer includes an oxidoreductase enzyme and a binder. The thickness ofthe first layer is selected so that when a read pulse is applied to thefirst and second electrodes during use, measurable species aresubstantially detected within the first layer and are not substantiallydetected external to the first layer.

In another aspect, a method of increasing the accuracy of quantitativeanalyte determination is provided. The method includes providing anelectrochemical sensor strip having at least one first layer includingan oxidoreductase enzyme, a mediator, and a binder. An analytecontaining sample is then introduced to the electrochemical sensor stripand an electric potential is applied in the form of a read pulse. Theduration of the read pulse substantially detects the ionized form of themediator within the first layer while substantially excluding fromdetection the ionized form of the mediator external to the first layer.

In one embodiment, an electrochemical sensor strip is provided,comprising: a base; a first electrode on the base, where the firstelectrode comprises at least one first layer on a first conductor, thefirst layer including a reagent layer; and a second electrode on thebase, the thickness of the first layer selected so that a read pulseapplied to the first and second electrodes during use substantiallydetects a measurable species within the first layer and substantiallydoes not detect the measurable species external to the first layer.

In another embodiment, the electrochemical sensor strip furthercomprises a second layer between the first conductor and the firstlayer, the thickness of the second layer selected so that a read pulseapplied to the first and second electrodes during use substantiallydetects the measurable species within the second layer and substantiallydoes not detect the measurable species external to the second layer,including the measurable species within the first layer, where thethickness of the first layer is not selected so that a read pulseapplied to the first and second electrodes during use substantiallydetects the measurable species within the first layer. The second layermay be at least 5 μm or from 8 to 25 μm thick. In another aspect, thesecond layer may be at least 1 μm or from 5 to 25 μm thick. The secondlayer may not include the oxidoreductase enzyme and/or a mediator, butmay include a polymeric material.

In another embodiment, a method of increasing the accuracy ofquantitative analyte determination is provided comprising: providing anelectrochemical sensor strip having a base, a first conductor on thebase, a second conductor on the base, and at least one first layer on atleast the first conductor, where the at least one first layer includes areagent layer including a binder; introducing an analyte containingsample having a liquid component to the electrochemical sensor strip,where the sample provides electrical communication between the first andsecond conductors; applying an electric potential between the first andsecond conductors in the form of a read pulse, the read pulse appliedfor a duration that substantially detects a measurable species withinthe first layer and substantially does not detect the measurable speciesexternal to the first layer; measuring the read pulse to provide aquantitative value of the analyte concentration in the sample withincreased accuracy in relation to an electrochemical sensor strip thatsubstantially detects the measurable species external to the firstlayer. An initial pulse and a time delay may be applied before the readpulse.

In another embodiment, an electrochemical sensor strip is provided,comprising: a base; a first electrode on the base, where the firstelectrode comprises at least one first layer on a first conductor, thefirst layer including a mediator, a binder, and at least one of glucoseoxidase, glucose dehydrogenase, and mixtures thereof; and a secondelectrode on the base comprising a soluble redox species, the solubleredox species including at least one of an organotransition metalcomplex, a transition metal coordination metal complex, and mixturesthereof, the thickness of the first layer selected so that a read pulseapplied to the first and second electrodes during use substantiallydetects a measurable species within the first layer and substantiallydoes not detect the measurable species external to the first layer. Thesecond electrode of the electrochemical sensor strip may comprise asecond redox species on a second conductor, where the soluble redoxspecies is a first redox species of a redox pair including the firstspecies and the second species, and where the molar ratio of the firstredox species to the second redox species is greater than about 1.2:1.

In another embodiment, an electrochemical sensor strip is provided,comprising: a base; a lid contacting the base to define a gap; a firstelectrode on the base including a first conductor; a second layer on thefirst conductor, where a reagent layer does not reside between the firstconductor and the second layer; a second electrode on the base; and areagent layer in the gap, the thickness of the second layer selected sothat a read pulse applied to the first and second electrodes during usesubstantially detects a measurable species within the second layer andsubstantially does not detect the measurable species external to thefirst layer.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention can be better understood with reference to the followingdrawings and description. The components in the figures are notnecessarily to scale, emphasis instead being placed upon illustratingthe principles of the invention. Moreover, in the figures, likereferences numerals generally designate corresponding parts throughoutthe different views.

FIG. 1 is a top view diagram of a sensor base containing a workingelectrode and a counter electrode.

FIG. 2 is an end view diagram of the sensor base of FIG. 1.

FIG. 3 is a top view diagram of a sensor base and electrodes under adielectric layer.

FIGS. 4-6 are top views of three electrode sensor strips.

FIG. 7 is an end view diagram of the sensor base of FIG. 5 depicting thethird electrode.

FIG. 8 is a perspective representation of a completely assembled sensorstrip.

FIGS. 9A and 9B depict a working electrode having a conductor surfaceand a DBL during the application of long and short read pulses.

FIGS. 10A and 10B are graphs illustrating the improvement in measurementaccuracy when a DBL is combined with a short read pulse in accord withthe present invention.

FIGS. 11A and 11B are graphs establishing the improvement in accuracyarising from a reduction in the duration of the read pulse when a DBL isutilized.

FIG. 12 is a table comparing the bias results for 1 and 10 second readpulses from multiple analyses performed with multiple types of sensorstrips having a diffusion barrier layer.

FIGS. 13A-13C are graphs illustrating the ability of a sensor striphaving a DBL in accordance with the present invention to accuratelymeasure the true glucose concentration of a sample utilizing a shortread pulse.

FIGS. 14A-14F are graphs illustrating the decay profiles for multipleglucose concentrations when different thicknesses of a combinationDBL/reagent layer were utilized with sequential 1 sec read pulses.

FIG. 14G illustrates the decay profiles for multiple glucoseconcentrations for a 1 to 2 μm combined DBL/reagent layer with aninitial 1 second pulse followed by sequential 0.25 second read pulses.

FIG. 15 compares the precision between sensor strips having a DBL andgap volumes of 1, 3, 5, and 10 mL when read pulses of 1, 5, 10, and 15seconds were applied.

DETAILED DESCRIPTION

Tiny electrochemical cells provide patients with the benefit of nearlyinstantaneous measurement of their glucose levels. One of the mainreasons for errors in these measurements is the hematocrit effect. Thehematocrit effect arises when red blood cells randomly affect thediffusion rate of measurable species to the conductor surface of theworking electrode.

By measuring a measurable species residing in a diffusion barrier layer(DBL), to the substantial exclusion of the measurable species residingexterior to the DBL, measurement errors introduced by the hematocriteffect and manufacturing variances may be reduced. Substantial exclusionof the measurable species external to the DBL may be achieved byselecting the thickness of the DBL on the basis of the read pulseduration or by selecting the duration of the read pulse on the basis ofthe thickness of the DBL.

FIG. 1 is a top view diagram of a sensor base 10 having conductors 12and 14 that contains a working electrode 20 and a counter electrode 30.FIG. 2 is an end view diagram of the sensor base 10, depicting theworking electrode 20 and the counter electrode 30. The working electrode20 may include a first main conductor 22, while the counter electrode 30may include a second main conductor 32. Optionally, surface conductors24 and 34 may reside on the main conductors 22 and 32, respectively. Adiffusion barrier layer (DBL) 28 also may reside on the main conductor22 of the working electrode 20.

In one aspect, the main conductors 22 and 32 may include metal foil thatis contiguous with the surface conductors 24 and 34 that include one ormore layers of conductive carbon powder. The working electrode 20 mayinclude a first reagent layer 26 residing on the first main conductor22, while the counter electrode 30 may include a second reagent layer 36residing on the second main conductor 32. In another aspect, the counterelectrode 30 may be a counter/reference electrode or a counter electrodecoated with a soluble redox species having a known oxidation orreduction potential. The sensor base 10 may have other configurations,including those with fewer or additional components as is known in theart. For additional sensor designs, see, for example, U.S. Pat. Nos.5,120,420 and 5,798,031, both of which are incorporated by reference.

The sensor base 10 is preferably an electrical insulator that mayisolate the electrochemical system from its surroundings. In use, theworking electrode 20 and the counter electrode 30 are in electricalcommunication with a measurement device (not shown) through theconductors 12 and 14, respectively. The measurement device may apply anelectrical potential between the working electrode 20 and the counterelectrode 30. The measurement device then may quantify the electricalcurrent flowing between the working electrode 20, a sample (not shown),and the counter electrode 30. The sample may establish electricalcommunication between the electrodes 20, 30.

The main conductors 22 and 32 and the optional surface conductors 24 and34 of electrodes 20 and 30 may contain any electrically conductivesubstance, including metals, conductive polymers, and conductive carbon.Examples of electrically conductive substances include a thin layer of ametal, such as gold, silver, platinum, palladium, copper, or tungsten,as well as a thin layer of conductive carbon powder. Preferably,conductors that are in contact with the sample during the use of thesensor are made of inert materials, such that the conductor does notundergo a net oxidation or a net reduction during the analysis. Morepreferably, electrodes that are in contact with the sample during theuse of the sensor are made of non-ionizing materials, such as carbon,gold, platinum, and palladium.

Metals may be deposited on the base 10 by deposition of a metal foil, bychemical vapor deposition, or by deposition of a slurry of the metal.Conductive carbon may be deposited on the base 10, for example, bypyrolysis of a carbon-containing material or by deposition of a slurryof carbon powder. The slurry may contain more than one type ofelectrically conductive substance. For example, the slurry may containboth palladium and carbon powder. In the case of slurry deposition, thefluid mixture may be applied as an ink to the base material, asdescribed in U.S. Pat. No. 5,798,031.

When the surface conductors 24 and 34 are deposited on the mainconductors 22 and 32, it is preferred that the substance from which thesurface conductors are made is a non-ionizing conductive material. Whenthe main conductors 22 and 32 are utilized without the distinct surfaceconductors 24 and 34, it is preferred that the conductive material fromwhich the main conductors are made is non-ionizing. More preferably, theportion of the counter electrode 30 in contact with the second reagentlayer 36 (either the main conductor 32 or the surface conductor 34) is anon-ionizing material.

A DBL may be integral to the reagent layer 26 or it may be a distinctlayer 28 as depicted in FIG. 2. Thus, the DBL may be formed as acombination reagent/diffusion barrier layer on the conductor surface, asa distinct layer on the conductor surface, as a distinct layer on theconductor surface on which the reagent layer resides, or as a distinctlayer on the reagent layer.

The DBL provides a porous space having an internal volume where ameasurable species may reside. The pores of the DBL are selected so thatthe measurable species may diffuse into the DBL, while physically largersample constituents, such as RBC, are substantially excluded. Althoughconventional sensor strips have used various materials to filter RBCfrom the working electrode, the DBL of the present inventionadditionally provides an internal porous space to contain and isolate aportion of the measurable species from the sample volume.

By controlling the length of the measurement reaction at the conductorsurface, the sensor strip may measure the measurable species internal tothe DBL, while substantially excluding from measurement the measurablespecies external to the DBL. In relation to the conductor surface, theinternal volume of the DBL alters the physical parameter of thediffusion rate of the measurable species it contains in relation to thediffusion rate of the measurable species outside of the DBL.

Because the measurable species internal to the DBL diffuses at adifferent rate to the conductor surface than the measurable speciesexternal to the DBL, the length of the measurement reaction at theworking electrode selects which measurable species is preferentiallymeasured. While identical from a molecular standpoint, the differentdiffusion rates of the measurable species internal and external to theDBL allow their substantial differentiation.

Because the reagent layer 26 of the working electrode 20 may include abinder, any portion of the binder that does not solubilize into thesample prior to the application of a read pulse can function as the DBL.When the reagents are combined with the binder material to provide bothsupport to the reagents and to provide the DBL, the binder material ispreferably a polymeric material that is at least partially watersoluble. In this manner, a portion of the binder material cansolubilize, while the remainder of the binder material may remain on themain conductor 22 to function as the DBL.

Suitable partially water soluble polymeric materials include, but arenot limited to, poly(ethylene oxide) (PEO), carboxy methyl cellulose(CMC), polyvinyl alcohol (PVA), hydroxyethyl cellulose (HEC),hydroxypropyl cellulose (HPC), methyl cellulose, ethyl cellulose, ethylhydroxyethyl cellulose, carboxymethyl ethyl cellulose, polyvinylpyrrolidone (PVP), polyamino acids such as polylysine, polystyrenesulfonate, gelatin, acrylic acid, methacrylic acid, starch, and maleicanhydride salts thereof, derivatives thereof, and combinations thereof.Among the above, PEO, PVA, CMC, and HEC are preferred at present, withCMC and PEO being especially preferred at present.

Materials that were conventionally used to form filters for theexclusion of RBC from the working electrode also may be used in the DBL.This may be achieved by increasing the thickness of the material or byreducing the length of the measurement reaction at the working electrodein relation to when the material was used as filter. It also may beaccomplished by forming the material in a manner that modifies itsviscosity, such as through the introduction of salts like sodium orpotassium chloride.

In another aspect, the DBL may be the distinct DBL 28. The distinctlayer 28 may have an average initial thickness of at least 5 μm,preferably, from 8 to 25 μm, and more preferably from 8 to 15 μm. Inanother aspect, the distinct layer 28 may have an average initialthickness of at least 1 μm or preferably from 5 to 25 μm. When the DBLis a distinct layer, it may be made from a partially soluble polymericmaterial, such as the same material utilized as a binder in the reagentlayer 26, but lacking the reagents. The distinct layer 28 may be anymaterial that provides the desired pore space, but that is partially orslowly soluble in water during use of the sensor.

Although not shown in the figures, when the DBL is the distinct layer 28the reagent layer 26 may not reside on the distinct layer 28. Instead,the reagent layer 26 may reside on any portion of the sensor strip thatallows the reagent to solubilize in the sample. For example, the reagentlayer 26 may reside on the sensor base 10 or on a lid 50, as discussedbelow with regard to FIG. 8.

In one aspect, the first and second reagent layers may include the sameconstituents and may reside on both the first and second main conductors22, 32. When the reagent layers 26 and 36 for the working and counterelectrodes, 20 and 30 respectively, have different compositions, thereagent layer for each electrode may be separately optimized. Thus, thefirst reagent layer 26 may contain ingredients that facilitate thereaction of the analyte and the communication of the results of thisreaction to the first main conductor 22.

Similarly, the second reagent layer 36 may contain ingredients thatfacilitate the free flow of electrons between the sample being analyzedand the second main conductor 32. For example, a soluble redox speciesincorporated into the second reagent layer 36 may undergo an oppositeredox reaction to the analyte. Even though the redox species is beingconsumed (i.e. converted into its counterpart species) during use, itmay be present in a high enough concentration in the second reagentlayer 36 to provide a relatively constant linear relationship betweenthe measured current and the analyte concentration for the time scale ofthe analysis. Thus, improved performance may be obtained by separatelyoptimizing the reagent layers 26 and 36 in comparison to sensor stripsutilizing the same reagent layer for both electrodes.

A large molar ratio of the soluble redox species placed on the counterelectrode 30 may increase the shelf life of the sensor strip. A smalldegree of spontaneous conversion of the soluble redox species into itscounterpart species can occur during the time between the manufacture ofthe strip and its use with a sample. Since the relative concentrationwill remain high due to the excess soluble redox species, the sensor mayproduce accurate results after storage.

The first reagent layer 26 residing on the first main conductor 22 mayinclude an oxidoreductase. The oxidoreductase may be specific for theanalyte of interest. The oxidoreductase may be specific for a substratesuch that the reaction of the oxidoreductase and its substrate isaffected by the presence or amount of the analyte of interest. While ina formal sense a substrate is affected by the amount of the analyte,unless stated otherwise, the term analyte is intended to include anactual analyte present in the sample or its substrate in thisdescription and the appended claims.

Examples of oxidoreductases and their specific analytes are given belowin Table I.

TABLE I Oxidoreductase (reagent layer) Substrate/analyte Glucosedehydrogenase β-glucose Glucose oxidase β-glucose Cholesterol esterase;cholesterol oxidase Cholesterol Lipoprotein lipase; glycerol kinase;Triglycerides glycerol-3-phosphate oxidase Lactate oxidase; lactatedehydrogenase; diaphorase Lactate Pyruvate oxidase Pyruvate Alcoholoxidase Alcohol Bilirubin oxidase Bilirubin Uricase Uric acidGlutathione reductase NAD(P)H Carbon monoxide oxidoreductase Carbonmonoxide

For example, an alcohol oxidase can be used in a reagent layer toprovide a sensor that is sensitive to the presence of alcohol in asample. Such a system could be useful in measuring blood alcoholconcentrations. In another example, glucose dehydrogenase or glucoseoxidase can be used in the reagent layer to provide a sensor that issensitive to the presence of glucose in a sample. This system could beuseful in measuring blood glucose concentrations, for example inpatients known or suspected to have diabetes. If the concentrations oftwo different substances are linked through a known relationship, thenthe measurement of one of the substances through its interaction withthe oxidoreductase can provide for the calculation of the concentrationof the other substance. For example, an oxidoreductase may provide asensor that is sensitive to a particular substrate, and the measuredconcentration of this substrate can then be used to calculate theconcentration of the analyte of interest.

The first reagent layer 26 may include a mediator. Without wishing to bebound by any theory of interpretation, it is believed that mediators mayact either as a redox cofactor in the initial enzymatic reaction or as aredox collector to accept electrons from or donate electrons to theenzyme or other species after the reaction with the analyte hasoccurred. In the situation of a redox cofactor, the mediator is believedto be the species that balances the redox reaction of the analyte. Thusif the analyte is reduced, the mediator is oxidized. In the situation ofa redox collector, another species may have been oxidized or reducedinitially to balance the redox reaction of the analyte. This species maybe the oxidoreductase itself, or it may be another species such as aredox cofactor.

Mediators in enzymatic electrochemical cells are described in U.S. Pat.No. 5,653,863, for example, which is incorporated herein by reference.In some cases, the mediator may function to regenerate theoxidoreductase. In one aspect, if the enzyme oxidizes an analyte, theenzyme itself is reduced. Interaction of this enzyme with a mediator canresult in reduction of the mediator, together with oxidation of theenzyme to its original, unreacted state. Interaction of the mediatorwith the working electrode 20 at an appropriate electrical potential canresult in a release of one or more electrons to the electrode togetherwith oxidation of the mediator to its original, unreacted state.

Examples of mediators include OTM and coordination complexes, includingferrocene compounds, such as 1,1′-dimethyl ferrocene; and includingcomplexes described in U.S. Pat. No. 5,653,863, such as ferrocyanide andferricyanide. Examples of mediators also include electro-active organicmolecules including coenzymes such as coenzyme pyrroloquinoline quinone(PQQ); the substituted benzoquinones and naphthoquinones disclosed inU.S. Pat. No. 4,746,607, which is incorporated herein by reference; theN-oxides, nitroso compounds, hydroxylamines and oxines specificallydisclosed in EP 0 354 441, which is incorporated herein by reference;the flavins, phenazines, phenothiazines, indophenols, substituted1,4-benzoquinones and indamines disclosed in EP 0 330 517, which isincorporated herein by reference; and the phenazinium and phenoxaziniumsalts disclosed in U.S. Pat. No. 3,791,988, which is incorporated hereinby reference. A review of electrochemical mediators of biological redoxsystems can be found in Analytica Clinica Acta. 140 (1982), pages 1-18.Examples of electro-active organic molecule mediators also include thosedescribed in U.S. Pat. No. 5,520,786, which is incorporated herein byreference, including 3-phenylimino-3H-phenothiazine (PIPT), and3-phenylimino-3H-phenoxazine (PIPO).

The second reagent layer 36 may include a soluble redox species. Thesoluble redox species undergoes the opposite redox reaction relative tothe reaction of the analyte of the oxidoreductase, and in so doing isconverted into its counterpart species of the redox pair. For example,if the analyte is reduced, the soluble redox species is oxidized; and ifthe analyte is oxidized, the soluble redox species is reduced. Thecounterpart species of the redox pair may also be present in the layer,but it is preferably present in a concentration lower than theconcentration of the primary redox species. More preferably, the redoxspecies in the reagent layer on the counter electrode is exclusively thesoluble redox species that undergoes the opposite reaction relative tothe reaction of the substrate of the oxidoreductase.

A soluble redox species may be an electro-active organic molecule, anorganotransition metal complex, a transition metal coordination complex,or a combination thereof. Suitable electro-active organic molecules mayinclude coenzyme pyrroloquinoline quinone (PQQ), substitutedbenzoquinones and naphthoquinones, N-oxides, nitroso compounds,hydroxylamines, oxines, flavins, phenazines, phenothiazines,indophenols, indamines, phenazinium salts, and phenoxazinium salts.

Suitable soluble redox species may also be OTM complexes or transitionmetal coordination complexes. Many transition metals occur naturally ascompounds with hydrogen, oxygen, sulfur, or other transition metals, andthese transition metals are generally observed in one or more oxidationstates. For example iron, chromium, and cobalt are typically found inoxidation states of +2 (i.e. II) or +3 (i.e. III). Thus, iron (II) andiron (III) are two species of a redox pair. Many elemental metals ormetal ions are only sparingly soluble in aqueous environments. This lackof solubility limits their utility as redox species in balancing theredox reactions in an electrochemical analysis system. The solubility ofthe otherwise sparingly soluble metals or metal ions may be improvedthrough bonding or coordination with ligands.

Typically, the metal in an organotransition metal complex or atransition metal coordination complex is the moiety in the complex thatis actually reduced or oxidized during use of the sensor strip. Forexample, the iron center in ferrocene [Fe(II)(C₅H₆)₂] and in theferrocyanide ion [Fe(II)(CN)₆]⁴⁻ is in the +2 formal oxidation state,while the ferricyanide ion [Fe(III)(CN)₆]³⁻ contains iron in its +3formal oxidation state. Together, ferrocyanide and ferricyanide togetherform a redox pair. Depending on the type of oxidoreductase present inthe reagent layer of the working electrode, either metal complex canfunction as the soluble redox species in the reagent layer on thecounter electrode. An example of a redox pair containing transitionmetal coordination complexes is the combination of two species ofruthenium hexaamine, [Ru(III)(NH₃)₆]³⁺ and [Ru(II)(NH₃)₆]²⁺.

The soluble redox species is capable of forming a redox pair during useof the electrochemical sensor strip. The species of this redox pair thatis present in the reagent layer 36 on the counter electrode 30, referredto as the first species, is preferably present in a greater molar amountthan its counterpart species (i.e. the second species) of the same redoxpair. Preferably, the molar ratio of the first species to the secondspecies is at least 1.2:1. More preferably, the molar ratio of the firstspecies to the second species is at least 2:1. Still more preferably,the molar ratio of the first species to the second species is at least10:1 or at least 100:1. In an aspect especially preferred at present,the second species of the redox pair is present in an amount of at most1 part per thousand (ppt) or at most 1 part per million (ppm), prior tothe use of the sensor strip in an analysis.

Preferably, the soluble redox species is solubilized in the sample andmixes with the analyte and other sample constituents. The soluble redoxspecies will, over time, mix with the enzyme and the mediator, althoughthis may not occur to any measurable degree over the course of theanalysis. The soluble redox species is not separated from the liquidsample by a mechanical barrier, nor is it separate from the liquidsample by virtue of its existence in a separate phase that is distinctfrom the liquid sample.

In a preferred embodiment, a soluble redox species is chosen having astandard reduction potential of +0.24 volts or greater, versus thestandard hydrogen electrode (SHE). In another preferred embodiment, asoluble redox species is chosen having a standard reduction potential of+0.35 volts or greater, versus SHE. In yet another preferred embodiment,a redox species having a reduction potential of about +0.48 volts versusSHE (in 0.01 M HCl) is chosen.

Thus, a wide variety of combinations of oxidoreductases, mediators, andsoluble redox species can be used to prepare an electrochemicalanalytical sensor. The use of soluble redox species having higher orlower oxidation numbers relative to their counterpart species in theredox pair is dictated by the type of reaction to be performed at theworking electrode.

In one example, the analyte undergoes oxidation by interaction with anoxidase or a dehydrogenase. In this case, the more concentrated redoxspecies on the counter electrode has the higher oxidation number. Aspecific example of this situation is the analysis of glucose usingglucose oxidase or glucose dehydrogenase. In another example, theanalyte undergoes reduction by interaction with a reductase. In thiscase, the more concentrated redox species on the counter electrode hasthe lower oxidation number. In either of these examples, the mediatormay be the same substance as the more concentrated redox species on thecounter electrode or the redox species of another redox pair.

FIG. 3 is a top view diagram of the sensor base 10, including theconductors 12 and 14 under a dielectric layer 40, and the electrodes 20and 30. The dielectric layer 40 may partially cover the electrodes 20and 30 and may be made from any suitable dielectric layer, such as aninsulating polymer. The dielectric layer 40 may isolate the portions ofthe electrodes that are in contact with the first and second reagentlayers 26 and 36 from the portions of the electrodes that are in contactwith the conductors 12 and 14. The dielectric layer 40, if present, maybe deposited on the sensor base 10 before, during, or after the coatingof the electrodes 20 and 30 with the reagent layers 26 and 36,respectively.

The electrodes 20, 30 may be coated with the reagent layers 26, 36 byany convenient means, such as printing, liquid deposition, or ink-jetdeposition. In one aspect, the reagent layer is deposited on theelectrodes 20, 30 by printing. With other factors being equal, the angleof the printing blade may inversely affect the thickness of the reagentlayer residing on the electrodes 20, 30. For example, when the blade ismoved at an approximately 82° angle to the sensor base 10, the resultingreagent layer or layers may have a thickness of approximately 10 μm.Similarly, when a blade angle of approximately 62° to the sensor base 10is utilized, a thicker 30 μm layer may be produced. In this aspect,lower blade angles may provide thicker reagent layers. In addition toblade angle, other factors affect the resulting thickness of the reagentlayers 26, 36, including the thickness of the material making up thereagent layer.

FIGS. 4-6 are top views of three electrode sensor strips, each havingthe sensor base 10, the working electrode 20, the counter electrode 30,the conductors 12 and 14, a conductor 13, and a third electrode 70. Thethird electrode 70 may be in electrical communication with themeasurement device (not shown) through the conductor 13.

The measurement device may measure an electric potential flowing betweenthe working electrode 20, the third electrode 70, and a sample (notshown) that establishes electrical communication between the electrodes.In another aspect, the measurement device may apply and measure anelectrical potential provided to the working electrode 20, the thirdelectrode 70, and the sample. The sensor base 10 may have otherconfigurations including those with fewer or additional components as isknown in the art.

FIG. 7 is an end view diagram of the sensor base 10 of FIG. 5 depictingthe optional third electrode 70. In one aspect, the optional thirdelectrode 70 may be a true reference electrode. In another aspect, thethird electrode 70 may be coated with a third reagent layer 76 includinga soluble redox species. An optional third electrode surface conductor74 may reside on a third main conductor 72. In one aspect, the thirdmain conductor 72 includes metal foil while the surface conductor 74includes one or more layers of conductive carbon powder. The thirdreagent layer 76 and the second reagent layer 36 may include the sameconstituents or have different constituents depending on the intendeduse. In one aspect, the third reagent layer 76 is a portion of thesecond reagent layer 36, which is deposited on the main conductors 32and 72.

When the surface conductor 74 is deposited on the main conductor 72, itis preferred that the substance from which the surface conductor is madeis a non-ionizing conductive material. When the main conductor 72 isutilized without the distinct surface conductor layer 74, it ispreferred that the conductive material from which the main conductor ismade is non-ionizing. More preferably, the portion of the thirdelectrode 70 in contact with the third reagent layer 76 (either the mainconductor 72 or the surface conductor 74) is a non-ionizing material.The third reagent layer 76 may include the same constituents as thefirst and second reagent layers 26 (not shown) and 36. In anotheraspect, the third reagent layer 76 may include the same constituents asthe second reagent layer 36. In yet another aspect, the third reagentlayer 76 may include ingredients that are specifically tailored toimprove the free flow of electrons between the sample being analyzed andthe third main conductor 72.

The reagent layer 76 may contain a soluble redox species as describedabove with regard to FIG. 2. Preferably the reagent layer 76 of thethird electrode 70 is identical in composition to the reagent layer 36of the counter electrode 30. If the reagent layers on the third andcounter electrodes are identical, then it may be desirable to coat bothelectrodes with a single portion of the reagent layer composition.

The use of the third electrode 70 may be desirable for someapplications. Increased accuracy in the applied voltage can provide forbetter accuracy in the measurement of the analyte. When using the thirdelectrode 70, it may also be possible to reduce the size of the counterelectrode 30 or to apply a smaller amount of the redox species to thecounter electrode. If the third electrode 70 is positioned upstream ofthe counter electrode 30, as illustrated in FIG. 6, then it may bepossible to detect when insufficient sample has been applied to thestrip, a situation referred to as “under-fill.” Under-fill detection mayoccur when there is sufficient sample to complete the circuit betweenthe working electrode 20 and the third electrode 70, but not to coverthe counter electrode 30. The lack of electrical current in the cell canbe converted electronically into a signal to the user, instructing theuser to add additional sample to the strip.

FIG. 8 is a perspective representation of an assembled sensor strip 800including the sensor base 10, at least partially covered by a lid 50that includes a vent 54, a concave area 52, and an input end opening 60.Preferably, the lid 50 covers, but does not contact, the reagent layers26 and 36 (not shown), thus providing a gap 56 between the lid 50 andthe electrodes.

A biological sample may be transferred to the electrodes by introducingthe liquid sample to the opening 60 of the sensor strip 800. The liquidfills the gap 56 while expelling the air previously contained by the gap56 through the vent 54. In this manner, the sample provides electricalcommunication between the electrodes. The gap 56 may contain a substance(not shown) that assists in retaining the liquid sample in the gap byimmobilizing the sample and its contents in the area above theelectrodes. Examples of such substances include water-swellablepolymers, such as carboxymethyl cellulose and polyethylene glycol; andporous polymer matrices, such as dextran and polyacrylamide.

If a sample introduced through the opening 60 contains an analyte forthe oxidoreductase, the redox reaction between the analyte and theenzyme can begin once the reagent layers and the sample are in contact.The electrons produced or consumed from the resultant redox reaction canbe quantified by applying an electrical potential (i.e. voltage) betweenthe working electrode and the counter electrode, and measuring thecurrent. This current measurement may be correlated with theconcentration of the analyte in the sample, provided the system has beencalibrated with similar samples containing known amounts of the analyte.

Alternatively, the third electrode 70 (FIGS. 4-7) may be used to monitorthe applied voltage. Any drift in the intended value of the electricalpotential may provide feedback to the circuitry through the thirdelectrode, so that the voltage can be adjusted appropriately. Ameasurement device preferably contains the necessary circuitry andmicroprocessors to provide useful information, such as the concentrationof the analyte in the sample, the concentration of the analyte in thebody of the patient, or the relevant concentration of another substancethat is related to the measured analyte.

Once the sample is introduced through the opening 60, the sample beginsto solubilize and react with the reagent layers 26, 36, and optionally76. It may be beneficial to provide an “incubation period” during whichthe reagents convert a portion of the analyte into a measurable speciesprior to the application of an electrical potential. While a longerincubation period may be utilized, preferably, a voltage is initiallyapplied to the sensor strip 800 at the same time as, or immediatelyafter, the introduction of the sample through the opening 60. A morein-depth treatment of incubation periods may be found in U.S. Pat. Nos.5,620,579 and 5,653,863.

The initially applied voltage may be maintained for a set time period,such as about 10 seconds for a conventional sensor strip, and thenstopped. Then no voltage may be applied for a set delay time period,such as about 10 seconds for a conventional sensor strip. After thisdelay time, a constant potential or a “read pulse” may be applied acrossthe working and counter electrodes of the sensor strip to measure theconcentration of the analyte. For conventional amperometric sensors,this read pulse is applied while the current is monitored for a readtime of from 5 to 10 seconds. Considering the sample volume contained bythe gap 56, a read pulse of from 5 to 10 seconds is relatively long.

In contrast to the conventional 5 to 10 second read pulse, when theworking electrode 20 (FIG. 2) is configured with the DBL of the presentinvention, shorter read times are preferred. FIGS. 9A and 9B depict aworking electrode 900 having a conductor surface 930 and a DBL 905during the application of long and short read pulses. A sample (notshown) is applied to the working electrode 900 and includes RBC 920residing on the DBL 905, external measurable species 910 residing in thesample, and internal measurable species 915 residing within the DBL 905.

As shown in FIG. 9A, when a long, 10 second read pulse is applied to theworking electrode 900, both the external and internal measurable species910 and 915 are measured at the surface of the conductor surface 930 bya change in oxidation state. During this measurement process, theexternal measurable species 910 diffuses through the sample region wherethe RBC 920 reside and through the DBL 905 to be measured at the surface930. As previously discussed, this diffusion of the external measurablespecies 910 through the RBC 920 during measurement introduces thehematocrit effect.

Furthermore, a long read pulse applied to a strip having a DBL, asdepicted in FIG. 9 a, performs similarly to a short read pulse appliedto a strip lacking a DBL. The similarity arises because measurablespecies diffuse through the RBC before being measured at the conductorsurface during the read pulse. In either instance, a substantial portionof the species measured during the read pulse originated in the testsample.

Unlike FIG. 9A, FIG. 9B represents the situation where a short readpulse is applied to the sensor strip 900 having the DBL 905 in accordwith the present invention. Here, the internal measurable species 915present in the DBL 905 undergoes a change in oxidation state at thesurface 930. Substantially all of the measurable species 910 residingexternal to the DBL 905 either remains external to the DBL or does notsubstantially diffuse through the DBL 905 to reach the conductor surface930 during the read pulse. Thus, the present invention substantiallyexcludes the external measurable species 910 from measurement, insteadmeasuring the measurable species 915 that is internal to the DBL 905.

FIGS. 10A and 10B are graphs illustrating the improvement in measurementaccuracy when a DBL is combined with a short read pulse in accord withthe present invention. Whole blood samples were combined withferrocyanide in a 5:1 dilution ratio to represent an underlying glucoseconcentration and measured with a 1 second read pulse. Thus, the initial20%, 40% and 60% hematocrit WB samples were diluted to 16%, 32% and 48%hematocrit (a 20% reduction of all three hematocrit values). The 20%,40%, and 60% lines represent the current measured for the blood samplescontaining 16%, 32%, and 48% hematocrit, respectively.

FIG. 10A shows the inaccuracies introduced by the hematocrit and othereffects from a bare conductor sensor strip lacking a DBL. The inaccuracyis represented as the difference between the 20% and 60% hematocritlines (the total hematocrit bias span) and represents the maximummeasurement inaccuracy attributable to the hematocrit effect. Smallerbias values represent a more accurate result. Similar performance wasobserved when a DBL was used with a longer read pulse as discussed abovewith regard to FIG. 9A.

Conversely, FIG. 10B shows a marked decrease in the distance between the20% and 60% calibration lines when a DBL in accordance with the presentinvention is combined with a 1 second read pulse. A distinct DBL of PEOpolymer and 10% KCl (without reagents) was printed on a conductorsurface as used for FIG. 10A above. Surprisingly, the total biashematocrit span with the DBL/short read pulse was nearly two-thirds lessthan the total bias span without the DBL. Thus, the present inventionsignificantly increased measurement accuracy in comparison to theconventional, bare conductor electrode.

While not wishing to be bound by any particular theory, it is presentlybelieved that by limiting the length of the read time with respect tothe thickness of the DBL, the present invention may exploit thephenomenon that the rate of diffusion of the measurable species into thepores of the DBL is varying, while the diffusion rate of the measurablespecies from the internal volume of the DBL to the surface of theconductor surface is constant. The varying degree of diffusion into theDBL caused by the WB matrix is believed to give rise to the hematocriteffect. Thus, measurement errors (bias) introduced by the sampleconstituents, including RBC, may be reduced by substantially limitingmeasurement to the measurable species present in the internal volume ofthe DBL, which are believed to have a relatively constant diffusionrate.

FIGS. 11A and 11B are graphs establishing the improvement in accuracyarising from a reduction in the duration of the read pulse when a DBL isutilized. FIG. 11A shows that without a DBL, the bias with read pulsesof 0.9, 5, 10, and 15 seconds are nearly identical. Regardless of thelength of the read pulse, the total bias span values are ˜40% and higher(50% on average), because the ability of the mediator to reach thesurface of the conductor is affected by the sample constituents,including RBC. However, as illustrated in FIG. 11B, when a DBL isutilized, the bias for the 0.9 second read pulse is generally less thanhalf of the bias observed for the 5 second read pulse and can be as muchas 2.5 times less than the bias observed for a conventional 10 secondread pulse, depending on the ferrocyanide concentration.

When combined with a DBL, read pulses of less than 5 seconds arepreferred and read pulses of less than 3 seconds are more preferred. Inanother aspect, read pulses from 0.1 to 2.8 or from 0.5 to 2.4 secondsare preferred. In yet another aspect, read pulses from 0.05 to 2.8 orfrom 0.1 to 2.0 seconds are preferred. At present, read pulses from 0.8to 2.2 or from 0.8 to 1.2 seconds are more preferred, while read pulsesfrom 0.1 to 1.5 or from 0.125 to 0.8 seconds are especially preferred.The thickness of the DBL present during application of the read pulsemay be selected so that during the pulse, the measurable speciesexternal to the DBL is substantially prevented from diffusing to thesurface of the conductor.

FIG. 12 is a table comparing the bias results for 1 and 10 second readpulses from multiple analyses performed with multiple types of sensorstrips having a DBL. The table shows the total bias span values for WBsamples containing 50, 100, 200, and 400 mg/dL glucose. Absolute biasvalues are listed for the 50 mg/dL samples, while % bias is shown forthe 100, 200, and 400 mg/dL samples. The bias values for the varyingglucose concentrations were averaged for both the 10 second and the 1second read pulses. The shorter 1 second read pulse provided asubstantial reduction in the bias values when compared with theconventional 10 second read pulse, with reductions from about 21% toabout 90%. For the 36 trials performed, the overall average biasreduction was about 50%. Thus, the combination of a DBL with a shortread pulse in accord with the present invention significantly increasedmeasurement accuracy.

FIGS. 13A-13C are graphs illustrating the ability of a sensor striphaving a DBL to accurately measure the true glucose concentration of asample utilizing a short read pulse. The data underlying the figures wascollected by measuring the current in WB and plasma solutions containingferrocyanide as the measurable species. Because the plasma samples lackRBC, the plasma measurements lack inaccuracies introduced by thehematocrit effect. Conversely, measurements taken in the WB samplesincluded inaccuracies introduced by the hematocrit effect.

FIG. 13A correlates plasma and WB measurements collected with a 1 secondread pulse for a bare conductor sensor strip. The slope of the resultingcorrelation plot is only 0.43, indicating that on average only 43% ofthe measurable species present in the WB samples was measured. Incomparison, FIG. 13B correlates plasma and WB measurements collectedwith a 1 second read pulse for a sensor strip having a DBL. The slope ofthe resulting correlation plot is a substantially higher 0.86,indicating that about 86% of the measurable species present in the WBsamples was measured. Thus, when compared with a bare conductor, a shortread pulse combined with a DBL in accordance with the present inventionmay provide a 100% improvement in the measured versus actual analyteconcentration in WB samples.

FIG. 13C illustrates that decreased duration read pulses enhancemeasurement performance for sensor strips equipped with a DBL in accordwith the present invention. The graph shows the correlation plots for 1,5, and 10 second read pulse measurements taken in the WB and plasmasamples previously described with respect to FIGS. 13A and 13B. The 1,5, and 10 second pulses have correlation plot slopes of 0.86, 0.78 and0.71, respectively. Thus, decreases in read pulse duration reducedmeasurement inaccuracies.

When a combination DBL/reagent layer is used, the length of the initialpulse and the delay affect the thickness of the DBL during the laterapplied read pulse. As previously discussed, combination DBL/reagentlayers rely on a water soluble binder material that is partiallysolubilized into the sample prior to application of the read pulse. Thereagent containing binder material remaining during the read pulseserves as the DBL.

Because solubilization of the binder material begins as soon as thesample is introduced through the opening 60 (FIG. 8), the time thatpasses during the initial pulse and delay periods affects how much ofthe combined layer remains on the conductor surface during the readpulse. Thus, shorter initial pulses and delay times may be preferred toensure that sufficient binder material remains on the conductor to serveas an effective DBL.

However, depending on the duration of the read pulse, a preferable upperlimit exists for the DBL thickness because an increased DBL thicknessmay result in a failure of the sensor system to reach “steady-state”before application of the read pulse. Before the sensor system reachessteady-state, the concentration of the measurable species in the DBLdoes not accurately represent the concentration of the measurablespecies in the sample. In one aspect, this discrepancy between theconcentrations of the measurable species in the DBL and the sample maybe attributed to the changing rehydration state of the DBL.

Thus, if read pulses are applied and recorded before the steady-statecondition is reached, the concentration of the measurable speciesmeasured may not correlate with that in the sample. This lack ofcorrelation between the measurable species concentration in the DBL andthe sample may introduce inaccuracies into the measurement, therebyoffsetting the accuracy improvement otherwise obtained by excluding themeasurable species external to the DBL from measurement.

FIGS. 14A through 14F present the results obtained for multiple glucoseconcentrations when different initial thicknesses of a combinationDBL/reagent layer were utilized with sequential 1 sec read pulses. Thedata was obtained utilizing multiple 200 mV read pulses, each of 1second duration, separated by 0.5 second waits. Table II below lists theapproximate average DBL/reagent layer thickness and the approximate timeto reach steady state for each figure. The approximate beginning of thesteady-state condition may be observed when the last in time data pointobtained for an individual read pulse represents the greatest currentvalue of the last in time data points acquired for any individual readpulse.

Thus, for FIG. 14F, the last in time (rightmost) ˜1750 nA data point forthe read pulse initiated at ˜1.5 seconds establishes that steady-statewas reached at about 2.5 seconds at the 674.8 mg/dl glucoseconcentration.

TABLE II Approximate DBL/ Approximate time reagent layer to reach steadyFIG. average thickness in μm. state in seconds. 14A 30 >10 14B 23 5.514C 16 4 14D 14 2.5 14E 12 2.5 14F 11 2.5 14G 1 to 2 1

The data in Table II establish that for a 1 second read pulse precededby a 0.5 second delay, the average initial thickness of a combinationDBL/reagent layer is preferably less than 30 or 23 micrometers (μm) andmore preferably less than 16 μm. Preferred average initial thicknessesof a combination DBL/reagent layer for use with a 0.5 to 5 second delayand a 0.5 to 1.2 second read pulse are from 5 to 15 μm or from 11 to 14μm. More preferred average initial thicknesses of a combinationDBL/reagent layer for use with a 0.5 to 5 second delay and a 0.05 to 2.8second read pulse are from 1 to 15 μm or from 2 to 5 μm. Thus, for a 0.8to 1.2 second read pulse, these thicknesses substantially excludemeasurable species external to the DBL from the conductor surface duringthe read pulse, while allowing the sensor system to reach asteady-state.

While the preferred initial thickness of the reagent layer applied tothe conductor is dependent on the initial pulse length, the delay time,and the duration of the read pulse, for read pulse durations of lessthan five seconds, reagent layer thicknesses of from 5 to 30 μm or from11 to 20 μm are preferred. Furthermore, for read pulses of 1.5 secondsor less in duration, reagent layer thicknesses of from 1 to 10 μm orfrom 2 to 5 μm are preferred. The desired average initial thickness of acombination DBL/reagent layer may be selected for a specific read pulselength, such as for the 1 second read pulse of Table II, on the basis ofwhen steady-state is reached.

In one aspect, initial pulse and delay times of less than 6 seconds arepreferred. Initial pulse times of from 1 to 4 seconds and delay times offrom 0.5 to 5 seconds are more preferred. In a preferred aspect, theinitial pulse and delay times are selected so that at least 50% of theaverage initial thickness of the combined DBL/reagent layer remains onthe conductor surface when the read pulse is applied. In another aspect,from 60 to 85% or from 70 to 80% of the average initial thickness of thecombined layer remains on the conductor surface when the read pulse isapplied.

The preferred thickness of the DBL for a specific read pulse length alsomay depend on the nature of the DBL. The slower the measurable speciesmoves through the DBL during measurement, the thinner the DBL required.However, if diffusion of the measurable species through the DBL is tooslow, it may be difficult to obtain the desired steady-state condition.The rate at which a measurable species diffuses through the DBL also maybe altered with additives that affect the ionic strength of the testsample and/or of the pore interiors of the DBL. In one aspect, theadditive may be a salt, such as sodium or potassium chloride, which ispresent in the deposition solution/paste at a 1 to 2 Molarconcentration. Other salts and compositions that affect the ionicstrength of the test sample as known to those of ordinary skill in theart of chemistry also may be used.

Another advantage of measuring the measurable species in the DBL with aless than 3 second read pulse is the reduction of measurementimprecision from varying sample volumes present in the gap 56 of thesensor strip 800 (FIG. 8). If a read pulse continues past the time whensubstantially all of the measurable species present in the gap 56 hasbeen measured, the measurement no longer represents the concentration ofmeasurable species in the sample, but is instead measuring the amount ofmeasurable species in the gap 56; a very different measurement. As theread pulse becomes long relative the volume of the gap 56, the currentmeasurement will depend on the volume of the gap 56, not the underlyinganalyte concentration. Thus, longer read pulses can result inmeasurements that are highly inaccurate with regard to analyteconcentration if the pulse length “overshoots” the measurable speciespresent in the gap 56.

Hence, any variance in the volume of the gap 56 present in theelectrochemical sensor strip may lead to measurement imprecision becausethe electronics in the measurement device apply the same potential andperform the same calculations for each test. Thus, for the same sample,a conventional sensor strip having a larger gap volume will show ahigher analyte concentration than a sensor strip having a smaller gapvolume if the read pulse overshoots the gap volume. By substantiallylimiting measurement to the measurable species present in the DBL, thepresent invention may reduce the imprecision introduced by sensor stripshaving different gap volumes. In this manner, the effect thatmanufacturing variability in the sensor strips would otherwise have onthe measurement results may be reduced.

FIG. 15 compares the precision between sensor strips having a DBL andgap volumes of 1, 3, 5, and 10 mL when read pulses of 1, 5, 10, and 15seconds were applied. Table A presents the data collected when a 2second pre-pulse and a 4 second delay was followed by read pulses of 1,5, 10, and 15 seconds. Table B presents the data collected when a 4second pre-pulse and a 2 second delay was followed by read pulses of 1,5, 10, and 15 seconds. Variances between slopes and intercepts of thecalibration lines for each combination of gap volume and read pulseduration are expressed as %-CV. The %-CV values from tables A and B showthat as the duration of the read pulse increases so does the imprecisionin the measurements due to the variation in the gap volume. For bothpulse sequences, the deviation between the gap volumes is least for the˜1 second read pulse, and largest for the 15 second read pulse. Theseresults further establish the benefit of utilizing a DBL with a shortpulse length in accord with the present invention.

In addition to the hematocrit effect and variances in gap volumes, whenthe measurable species present in the gap 56 (FIG. 8) is measured,positive errors in the analyte reading may be introduced if the liquidsample moves during the measurement. This movement of the sample canintroduce fresh analyte to the region around the working electrode wherea constant diffusion pattern was already in place, thus skewing themeasurement. By measuring the measurable species internal to the DBL,which has a relatively constant diffusion rate, with a short read pulse,while substantially excluding from measurement the varying diffusionrate measurable species external to the DBL, the present invention mayfurther reduce measurement errors introduced by movement of the sample.

In order to provide a clear and consistent understanding of thespecification and claims, the following definitions are provided.

The term “system” is defined as an electrochemical sensor strip inelectrical communication through its conductors with an electronicmeasurement device, which allows for the quantification of an analyte ina sample.

The term “measurement device” is defined as an electronic device thatcan apply an electric potential to the conductors of an electrochemicalsensor strip and measure the subsequent electrical currents. Themeasurement device also may include the processing capability todetermine the presence and/or concentration of one or more analytes inresponse to the measured electric potential.

The term “sample” is defined as a composition containing an unknownamount of the analyte of interest. Typically, a sample forelectrochemical analysis is in liquid form, and preferably the sample isan aqueous mixture. A sample may be a biological sample, such as blood,urine or saliva. A sample may be a derivative of a biological sample,such as an extract, a dilution, a filtrate, or a reconstitutedprecipitate.

The term “analyte” is defined as one or more substances present in thesample. The measurement process determines the presence, amount,quantity, or concentration of the analyte present in the sample. Ananalyte may interact with an enzyme or other species that is presentduring the analysis.

The term “accuracy” is defined as how close the amount of analytemeasured by a sensor strip corresponds to the true amount of analyte inthe sample.

The term “precision” is defined as how close multiple analytemeasurements are for the same sample.

The term “conductor” is defined as an electrically conductive substancethat remains stationary during an electrochemical analysis. Examples ofconductor materials include solid metals, metal pastes, conductivecarbon, conductive carbon pastes, and conductive polymers.

The term “non-ionizing material” is defined as a material that does notionize during the electrochemical analysis of an analyte. Examples ofnon-ionizing materials include carbon, gold, platinum and palladium.

The term “measurable species” is defined as any electrochemically activespecies that may be oxidized or reduced under an appropriate potentialat the electrode surface of an electrochemical sensor strip. Examples ofmeasurable species include an analyte, a substrate, or a mediator.

The term “steady-state” is defined as when the rate of diffusion of themeasurable species into the DBL is substantially constant.

The term “oxidoreductase” is defined as any enzyme that facilitates theoxidation or reduction of a measurable species. An oxidoreductase is areagent. The term oxidoreductase includes “oxidases,” which facilitateoxidation reactions where molecular oxygen is the electron acceptor;“reductases,” which facilitate reduction reactions where the analyte isreduced and molecular oxygen is not the analyte; and “dehydrogenases,”which facilitate oxidation reactions in which molecular oxygen is notthe electron acceptor. See, for example, Oxford Dictionary ofBiochemistry and Molecular Biology, Revised Edition, A. D. Smith, Ed.,New York: Oxford University Press (1997) pp. 161, 476, 477, and 560.

The term “mediator” is defined as a substance that can be oxidized orreduced and that can transfer one or more electrons between a firstsubstance and a second substance. A mediator is a reagent in anelectrochemical analysis and is not the analyte of interest, butprovides for the indirect measurement of the analyte. In a simplisticsystem, the mediator undergoes a redox reaction with the oxidoreductaseafter the oxidoreductase has been reduced or oxidized through itscontact with an appropriate analyte or substrate. This oxidized orreduced mediator then undergoes the opposite reaction at the workingelectrode and is regenerated to its original oxidation number.

The term “electro-active organic molecule” is defined as an organicmolecule that does not contain a metal and that is capable of undergoingan oxidation or reduction reaction. Electro-active organic molecules canbehave as redox species and as mediators. Examples of electro-activeorganic molecules include coenzyme pyrroloquinoline quinone (PQQ),benzoquinones and naphthoquinones, N-oxides, nitroso compounds,hydroxylamines, oxines, flavins, phenazines, phenothiazines,indophenols, and indamines.

The term “binder” is defined as a material that is chemically compatiblewith the reagents utilized in the reagent layer of the working electrodeand that provides physical support to the reagents, while containing thereagents on the electrode conductor.

The term “average initial thickness” refers to the average height of alayer in its dry state prior to introduction of a liquid sample. Theterm average is used because the top surface of the layer is uneven,having peaks and valleys.

The term “redox reaction” is defined as a chemical reaction between twospecies involving the transfer of at least one electron from a firstspecies to a second species. Thus, a redox reaction includes anoxidation and a reduction. The oxidation portion of the reactioninvolves the loss of at least one electron by the first species, and thereduction portion involves the addition of at least one electron to thesecond species. The ionic charge of a species that is oxidized is mademore positive by an amount equal to the number of electrons transferred.Likewise, the ionic charge of a species that is reduced is made lesspositive by an amount equal to the number of electrons transferred.

The term “oxidation number” is defined as the formal ionic charge of achemical species, such as an atom. A higher oxidation number, such as(III), is more positive, and a lower oxidation number, such as (II), isless positive. A neutral species has an ionic charge of zero (0). Theoxidation of a species results in an increase in the oxidation number ofthat species, and reduction of a species results in a decrease in theoxidation number of that species.

The term “redox pair” is defined as two conjugate species of a chemicalsubstance having different oxidation numbers. Reduction of the specieshaving the higher oxidation number produces the species having the loweroxidation number. Alternatively, oxidation of the species having thelower oxidation number produces the species having the higher oxidationnumber.

The term “oxidizable species” is defined as the species of a redox pairhaving the lower oxidation number, and which is thus capable of beingoxidized into the species having the higher oxidation number. Likewise,the term “reducible species” is defined as the species of a redox pairhaving the higher oxidation number, and which is thus capable of beingreduced into the species having the lower oxidation number.

The term “soluble redox species” is defined as a substance that iscapable of undergoing oxidation or reduction and that is soluble inwater (pH 7, 25° C.) at a level of at least 1.0 grams per Liter. Solubleredox species include electro-active organic molecules, organotransitionmetal complexes, and transition metal coordination complexes. The term“soluble redox species” excludes elemental metals and lone metal ions,especially those that are insoluble or sparingly soluble in water.

The term “organotransition metal complex,” also referred to as “OTMcomplex,” is defined as a complex where a transition metal is bonded toat least one carbon atom through a sigma bond (formal charge of −1 onthe carbon atom sigma bonded to the transition metal) or a pi bond(formal charge of 0 on the carbon atoms pi bonded to the transitionmetal). For example, ferrocene is an OTM complex with twocyclopentadienyl (Cp) rings, each bonded through its five carbon atomsto an iron center by two pi bonds and one sigma bond. Another example ofan OTM complex is ferricyanide (III) and its reduced ferrocyanide (II)counterpart, where six cyano ligands (formal charge of −1 on each of the6 ligands) are sigma bonded to an iron center through the carbon atomsof the cyano groups.

The term “coordination complex” is defined as a complex havingwell-defined coordination geometry, such as octahedral or square planargeometry. Unlike OTM complexes, which are defined by their bonding,coordination complexes are defined by their geometry. Thus, coordinationcomplexes may be OTM complexes (such as the previously mentionedferricyanide), or complexes where non-metal atoms other than carbon,such as heteroatoms including nitrogen, sulfur, oxygen, and phosphorous,are datively bonded to the transition metal center. For example,ruthenium hexaamine is a coordination complex having a well-definedoctahedral geometry where six NH₃ ligands (formal charge of 0 on each ofthe 6 ligands) are datively bonded to the ruthenium center. A morecomplete discussion of organotransition metal complexes, coordinationcomplexes, and transition metal bonding may be found in Collman et al.,Principles and Applications of Organotransition Metal Chemistry (1987)and Miessler & Tarr, Inorganic Chemistry (1991).

The term “on” is defined as “above” and is relative to the orientationbeing described. For example, if a first element is deposited over atleast a portion of a second element, the first element is said to be“deposited on” the second. In another example, if a first element ispresent above at least a portion of a second element, the first elementis said to be “on” the second. The use of the term “on” does not excludethe presence of substances between the upper and lower elements beingdescribed. For example, a first element may have a coating over its topsurface, yet a second element over at least a portion of the firstelement and its top coating can be described as “on” the first element.Thus, the use of the term “on” may or may not mean that the two elementsbeing related are in physical contact with each other.

While various embodiments of the invention have been described, it willbe apparent to those of ordinary skill in the art that other embodimentsand implementations are possible within the scope of the invention.Accordingly, the invention is not to be restricted except in light ofthe attached claims and their equivalents.

What is claimed is:
 1. An electrochemical sensor strip, comprising: abase; a first electrode on the base, the first electrode having a firstlayer on a first conductor, the first layer including a reagent layerand a second layer between the first conductor and the first layer, thesecond layer comprising an at least partially water-soluble polymericmaterial; and a second electrode on the base.
 2. The electrochemicalsensor strip of claim 1, where an average initial thickness of thesecond layer is at least about 1 μm.
 3. The electrochemical sensor stripof claim 1, where an average initial thickness of the second layer isfrom about 5 μm to about 25 μm.
 4. The electrochemical sensor strip ofclaim 1, where the first layer includes an oxidoreductase enzyme and amediator.
 5. The electrochemical sensor strip of claim 4, where thereagent layer comprises a binder connecting the oxidoreductase enzymeand the mediator.
 6. The electrochemical sensor strip of claim 1, wherethe second layer does not include a substantial amount of at least oneof an oxidoreductase enzyme and a mediator.
 7. The electrochemicalsensor strip of claim 1, where the first layer comprises a binderforming a diffusion barrier layer.
 8. The electrochemical sensor stripof claim 1, where the second electrode comprises the first layer on asecond conductor.
 9. An electrochemical sensor strip, comprising: afirst electrode on the base, the first electrode having a first layer ona first conductor, the first layer including a reagent layer and asecond layer between the first conductor and the first layer, the secondlayer including a polymeric material which comprises at least one ofpoly(ethylene oxide), carboxy methyl cellulose, hydroxyethyl cellulose,hydroxypropyl cellulose, polyvinyl alcohol, polyvinyl pyrrolidone,derivatives thereof, and combinations thereof; and a second electrode onthe base.
 10. A method of quantitative analyte determination with anelectrochemical sensor strip, the method comprising: receiving ananalyte-containing sample by the electrochemical sensor strip, theanalyte-containing sample having a liquid component, the electrochemicalsensor strip comprising a base with a first layer, a first conductor,and a second conductor on the base, the first layer including a reagentlayer and a second layer between the first conductor and the firstlayer, the second layer comprising an at least partially water-solublepolymeric material; applying a read pulse between the first conductorand the second conductor on the base of the electrochemical sensorstrip, the read pulse applied for a duration that substantially detectsa measurable species within the first layer on the first conductor; andmeasuring a quantitative value of an analyte concentration in theanalyte-containing sample in response to the read pulse.
 11. The methodof claim 10, where the read pulse comprises an electric potential. 12.The method of claim 10, where the analyte-containing sample resides in agap formed between the base and a lid of the electrochemical sensorstrip, the gap having a volume of about 1 mL to about 10 mL.
 13. Themethod of claim 10, further comprising applying an initial pulse and atime delay before the read pulse.
 14. The method of claim 13, where theinitial pulse is from about 1 second to about 4 seconds.
 15. The methodof claim 13, where the time delay is from about 2 seconds to about 4seconds.
 16. The method of claim 10, where the first layer includes anoxidoreductase enzyme, a mediator, and a binder connecting theoxidoreductase enzyme and the mediator.
 17. The method of claim 10,where the first layer comprises a binder forming a diffusion barrierlayer.
 18. The method of claim 10, where an average initial thickness ofthe second layer is at least about 1 μm.
 19. The method of claim 10,where an average initial thickness of the second layer is about 5 μm toabout 25 μm.
 20. A method of quantitative analyte determination with anelectrochemical sensor strip, the method comprising: receiving ananalyte-containing sample by the electrochemical sensor strip, theanalyte-containing sample having a liquid component, the electrochemicalsensor strip comprising a base with a first layer, a first conductor,and a second conductor on the base, the first layer including a reagentlayer and a second layer between the first conductor and the firstlayer, the second layer comprising at least one of poly(ethylene oxide),carboxy methyl cellulose, hydroxyethyl cellulose, hydroxypropylcellulose, polyvinyl alcohol, polyvinyl pyrrolidone, derivativesthereof, and combinations thereof; applying a read pulse between thefirst conductor and the second conductor of the electrochemical sensorstrip, the read pulse applied for a duration that substantially detectsa measurable species within the first layer on the first conductor; andmeasuring a quantitative value of an analyte concentration in theanalyte-containing sample in response to the read pulse.